Primary open angle glaucoma is a common, insidious ophthalmic disorder characterized by an increased resistance to outflow of aqueous humor. Extracellular matrix (ECM) has been implicated as a major component of the barrier outflow. Trabecular cells situated on the trabecular beams and juxtacanalicular cells secrete ECM components which are thought to impede outflow and maintain a normal intraocular pressure of approximately 7 mm above venous pressure. The purpose of this proposal is to study signals involved in the regulation of trabecular cells with respect to their production of specific ECM components. An understanding of how trabecular cell ECM production is modulated will lead to better therapies for primary open angle glaucoma and primary congenital glaucoma. The primary hypothesis of this proposal is that regulators exist which modify the production and turnover of ECM glycoproteins in the trabecular meshwork just as in many other biological systems. Regulators which will be studied include peptide growth factors, cytokines and monokines and the aqueous humor itself. The regulators to be studied initially include heparin binding growth factor type-2, epidermal growth factor and aqueous humor. Heparin binding growth factor has ben found to profoundly increase the production of ECM in an organ culture system used to study the trabecular meshwork. This proposal seeks to define the specific ECM components which are altered as well as to explore what specific alterations and modifications of trabecular ECM can be produced by other growth factors and cytokines. The endpoint substances to be evaluated are primarily glycoproteins and include heparin sulfate proteoglycan, collagens, laminin and fibronectin. Immunohistochemistry is used for localization, but the primary techniques are quantitative. Autoradiography, enzyme immunoassays, gel electrophoresis, in situ hybridization with cDNA probes from a colloidal gold immunolabeling technique will provide a quantitative approach using tissue from human cell and organ cultures. In preliminary work, innovative methods have been developed, including enhanced methods for light immunohistochemistry which preserve antigenicity of the trabecular meshwork. A new type of organ culture is described which circumvents some of the limitations of previous systems. This proposal is unique in its complimentary use of a trabecular cell culture system and two organ culture systems to define growth factor effects upon ECM. Further, it is unique in that comparative studies of factor effects upon ECM are both qualitative and quantitative. To date, little work has been done on the regulation of trabecular ECM. Using growth factors with a range of methods will add valuable insights into the behavior of the cells of the trabecular meshwork.